Stanniocalcin 2: characterization of the protein and its localization to human pancreatic alpha cells.

Stanniocalcin (STC) is a hormone that was originally identified in fish, where it inhibits calcium uptake by the gills and gut and stimulates phosphate adsorption by the kidney. Recently, two mammalian homologues of stanniocalcin were identified. The first (STC1) shows 61% identity to the fish stanniocalcins and appears to have a function similar to that of the fish stanniocalcins. The second homologue (STC2) is 30-38% identical to the fish stanniocalcins, and is characterized by unique cysteine and histidine motifs that are not found in the other stanniocalcins. We purified both the native hamster and recombinant human STC2 proteins and obtained a partial amino acid sequence of the hamster protein. Both proteins behave as a disulfide bonded homodimer, which undergoes post-translational modification(s). The STC2 gene was localized to human chromosome 5q35. Northern blot analysis revealed that the primary site of human STC2 production is the pancreas, and immunostaining localized the STC2 protein to a subpopulation of cells in the islet. Double immunostaining for STC2 and either insulin or glucagon revealed that STC2 protein is found in the alpha cells, but not the beta cells. We speculate that STC2 may play a role in glucose homeostasis.

A novel method for quantitative analysis of recombinant secreted proteins using two-dimensional electrophoresis.

We describe a two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-based approach for detecting and quantifying secreted recombinant proteins in media conditioned by baby hamster kidney (BHK) cells. Seven secreted proteins were analyzed in this system: leptin, thrombopoietin, thrombin, glycoprotein 130, soluble interleukin-2 receptor, and two novel sequences obtained from sequence database searches. BHK cells transfected with plasmids encoding each of these proteins, and cells transfected with empty plasmids (control cells), were metabolically labeled and the resulting conditioned media were analyzed by 2-D PAGE. Gel images derived from cells expressing recombinant proteins were compared with images from control cells in order to identify spots corresponding to the expressed proteins. All seven of the test proteins were successfully detected using this method. The sensitivity of the system was evaluated by diluting samples derived from high-expressing clones with conditioned media from control cells. The sensitivity of detection was protein-dependent, but recombinant proteins expressed at levels as low as 10 ng/mL could be detected. Quantification of recombinant protein levels was achieved by measuring spot intensities using phosphorimager analysis. The intensity of spots corresponding to recombinant proteins were compared with the spot intensity of an endogenous BHK protein which had been calibrated to a known standard. Estimates of the levels of expressed protein determined using this technique correlated with the levels determined using standard affinity assays. We conclude that this system provides a reliable method for quantifying levels of protein expression when specific assays are unavailable.

Specific phosphorylation of a site in the full-length form of the alpha 1 subunit of the cardiac L-type calcium channel by adenosine 3',5'-cyclic monophosphate-dependent protein kinase.

Voltage-gated L-type Ca2+ channels mediate Ca2+ entry into cells in response to membrane depolarization. Ca2+ entry through the cardiac Ca2+ channel determines the rate and force of contraction, and modulation of Ca2+ channel activity by beta-adrenergic agents acting through adenosine 3',5'-cyclic monophosphate-(cAMP)-dependent protein phosphorylation contributes to physiological regulation of cardiac function by the sympathetic nervous system. Immunoblotting experiments using site-directed anti-peptide antibodies against different peptide segments indicate that the alpha 1 subunit of the cardiac L-type Ca2+ channel exists in two size forms with apparent molecular masses of 240 and 210 kDa, which we call alpha 1(242) and alpha 1(210), Alpha 1(242) corresponds to the full-length cardiac alpha 1 subunit predicted from its cDNA sequence, while alpha 1(210) is truncated at its COOH terminus. Only alpha 1(242) is phosphorylated in vitro by cAMP-dependent protein kinase. Protein microsequencing and peptide mapping of wild-type and mutant fusion proteins show that this phosphorylation occurs at serine 1928 near the COOH terminus. Phosphorylation of this residue can be detected by phosphospecific antibodies raised against the corresponding phosphopeptide. Experiments with these antibodies show that alpha 1(242) is phosphorylated in intact cells expressing the cardiac alpha 1 subunit in response to increased intracellular levels of cAMP. These results identify serine 1928 on the alpha 1 subunit as a possible site of regulation by cAMP-dependent phosphorylation.

Differential proteolysis of the full-length form of the L-type calcium channel alpha 1 subunit by calpain.

This study examines the proteolysis of the carboxy terminal domain of the full-length (alpha 1(212)) and truncated (alpha 1(190)) forms of the rabbit skeletal muscle L-type calcium channel alpha 1 subunit by calpain I and calpain II. Although both forms of the alpha 1 subunit show little sensitivity to proteolysis by calpain II, alpha 1(212) is relatively more sensitive than alpha 1(190) to digestion by calpain I, the form of the enzyme regulated by micromolar concentrations of calcium. Calpain I cleaves a 37-kDa fragment from the C-terminus of alpha 1(212) in a time- and concentration-dependent manner and proteolysis is independent of the alpha 1(212) phosphorylation state. This proteolytic cleavage removes the major site of cyclic AMP-dependent phosphorylation from alpha 1(212) and may provide a mechanism for modifying the cyclic AMP-dependent regulation of L-type calcium channels in skeletal muscle.

Cardiac L-type Ca2+ channel triggers transmitter release in PC12 cells.

Among the various voltage-sensitive Ca2+ channels present in PC12 cells are the dihydropyridine (DHP)-sensitive L-channel, the omega-conotoxin (omega-CgTx)-sensitive N-channel, and an atypical omega-CgTx/DHP-insensitive Ca2+ channel. Depolarization-evoked Ca2+ entry and [3H]dopamine release is mediated by L-type Ca2+ channels determined by the use of Ca2+ channel antagonists, and a single protein of 250 kDa is recognized by L-type-specific antibodies. Screening of a PC12 cDNA library revealed two types of Ca2+ channels which were identified by partial sequencing. A pc12-L clone displayed virtually identical sequence homology to the cardiac L-type channel. The identical sequence homology of the single alternative splicing region confirmed clone pc12-L as the rbC-I transcript, a cardiac-neuronal alpha 1 subunit expressed in rat brain. Clone pc12-N displayed identical sequence homology to rbB-I, a neuronal alpha 1 subunit of the N-type Ca2+ channel expressed in rat brain; Northern blot analysis identified RNA of a size similar to that previously described for rat brain.

Characterization of the two size forms of the alpha 1 subunit of skeletal muscle L-type calcium channels.

The molecular properties of two size forms of the alpha 1 subunit of purified skeletal muscle calcium channels were analyzed. The minor, full-length, form, alpha 1(212), was found to have an apparent molecular mass of 214 kDa by Ferguson plot analysis, while the major, truncated, form, now designated alpha 1(190), had an apparent molecular mass of 193 kDa. Antibody mapping of the C-terminal region of alpha 1(190) with 10 anti-peptide antibodies placed the C terminus between residues 1685 and 1699. Three consensus sites for cAMP-dependent protein phosphorylation are present in the C-terminal region of alpha 1(212) but not in alpha 1(190), and they may be important for the regulation of the ion conductance activity of the calcium channel.

Subunits of purified calcium channels. Alpha 2 and delta are encoded by the same gene.

Polyacrylamide gel electrophoresis of purified rabbit skeletal muscle L-type calcium channel before and after reduction of disulfide bonds confirmed that 27- and 24-kDa forms of the delta subunit are disulfide-linked to the 143-kDa alpha 2 subunit. The amino acid sequences of three peptides obtained by tryptic digestion of the delta subunits corresponded to amino acid sequences predicted from the 3' region of the mRNA encoding alpha 2. One of these peptides had the same sequence as the N terminus of the 24- and 27-kDa forms of the delta subunit and corresponded to residues 935-946 of the predicted alpha 2 primary sequence. Anti-peptide antibodies directed to regions on the N-terminal side of this site recognized the 143-kDa alpha 2 subunit in immunoblots of purified calcium channels under reducing conditions, whereas an antipeptide antibody directed toward a sequence on the C-terminal side of this site recognized 24- and 27-kDa forms of the delta subunit. A similar result was obtained after immunoblotting using purified transverse tubules or crude microsomal membrane preparations indicating that alpha 2 and delta occur as distinct disulfide-linked polypeptides in skeletal muscle membranes. Thus, the delta subunits are encoded by the same gene as the alpha 2 subunit and are integral components of the skeletal muscle calcium channel.

Comparative inhibition profiles of human brain and mouse liver L-hexonate dehydrogenase.

The inhibition profiles of mouse liver and human brain hexonate dehydrogenase were compared. In general, the pattern for fluoride, lithium, phenobarbital, hydroxylamine and iodoacetate inhibition is similar. Contrary to previous findings, the mouse liver enzyme is potently inhibited by cupric and mercuric ions in sub-millimolar concentrations. The human brain enzyme is also inhibited by these cations. Inhibition of both enzymes by thiol-blocking agents (p-chloromercuribenzoate, iodoacetate) and enzyme protection by the first substrate NADPH but not by the second substrate, glucuronate suggests that both enzymes contain thiol groups essential for catalytic activity.